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Sheer Numbers

A team of microbiology researchers at Oregon State University has discovered several new groups of ocean microbes during the past 12 years. One of these recently discovered groups, the "SAR11" bacteria, may turn out to be one of the smallest, yet most abundant, life forms on Earth.

A mere teaspoon of seawater contains about a million bacteria cells. If all the bacteria in the ocean were added together, they would make up more biomass than all the fish and whales in the world’s oceans combined, according to OSU microbiologist Steve Giovannoni.

On the basis of sheer numbers alone, bacteria are staggeringly important to global food chains.

But much about SAR11 and other newly described ocean bacteria remains a mystery. Though they have been identified using genetic techniques, they are not yet cultured in the laboratory.

What are these superabundant, but elusive microbes doing out there? What do they consume? Excrete? How do they influence the Earth?

Questions such as these drive Giovannoni’s innovative research program at OSU called the "Laboratory for the Isolation of Novel Species" (LIONS) to uncover more about the biology and global role of little-understood ocean bacteria by culturing them in the laboratory.

How could such a prevalent group of microorganisms lie undiscovered for so long? Why did it take scientists until the early 1990s to find these microorganisms?

The story begins about 15 years ago, with the dawn of DNA technology, explained Giovannoni.

In the late 1980s, he and other microbiologists were suddenly armed with new techniques that could identify microbes by detecting bits of their genetic code without first growing the microbes in the lab.

"Scientists were aware of huge discrepancies between the number of cells they could count in nature and the number they could grow in the lab," said Giovannoni. "Some of us began to realize that a lot of bacteria just didn’t grow in a petri dish. We realized we just didn’t know a lot about many of them.

"Were the cells that grew in dishes representative of the bacteria that were abundant in nature? Or were there new organisms out there that we didn’t know how to grow?"

The earth's vast oceans are teeming with microscopic organisms like bacteria. Photo: Stone

Rather than having to capture and raise live cells in the lab to identify a new microbe, researchers could take a sample from any environment and analyze it for the presence and sequence of genetic code. It didn’t matter if an organism was dead or alive in any given sample. DNA or RNA would leave a unique, identifiable "fingerprint" for any given organism. Using the new techniques, scientists began to find evidence for life where life was previously thought to be impossible. One-celled organisms were turning up in rock layers far underground, in scalding hot springs and in mantle-heated ocean trenches miles below the surface.

The floor dropped out of the discipline of microbiology as it became evident to many, including Giovannoni, that the vast majority of microbes on Earth were yet to be discovered.

Microbiologist Steve Giovannoni, director of OSU's Laboratory for the Isolation of Novel Species, studies microbes in seawater collected from the open ocean. Photo: Lynn Ketchum

"In the past 12 to 14 years, there’s been a big change in how the average microbiologist looks at microbial diversity," said Giovannoni. "Before, not many microbiologists thought that microbial diversity or studying unculturable microbes had much potential for discovery.

"Now, scientists pretty much agree that the majority of microbes in the world have not been cultured. We know relatively little about most of them. Out of 1,000 kinds of microbes, 999 perish before they are detected by normal microbiological culturing methods."

In the late 1980s and 1990s, teams around the world began searching the world’s rocks, soils, waters and other extreme environments such as hot springs, deserts and ice caps for new organisms using DNA technology. Giovannoni turned to the world’s open oceans, which were thought to have few types of microorganisms.

"I started thinking about the kinds of mysteries that might be right under our nose," he said. "The open ocean is the most common habitat on the planet, yet we know so very little about it. We became gene hunters. In place of the organisms themselves, we began to hunt for their DNA. Their genes would tell us they are out there."

In 1989, soon after starting his appointment as an assistant professor at OSU, Giovannoni headed out the Sargasso Sea off Bermuda to hunt for new microbes in samples of seawater.

"The Sargasso Sea is a good place to study microbes because it is typical of open ocean conditions in many parts of the world," he explained. "The surface layers are low in nutrients. The warm water keeps the colder water and nutrients down near the bottom."

For the first time, microbiologists found the open ocean to be teeming with microbes that were previously unknown to exist.
Over the past decade, Giovannoni and his colleagues have analyzed data from seawater samples collected from the Sargasso, the North Atlantic, the Pacific, in the Santa Barbara Channel and off the Oregon Coast. With each sample, billions of microbes were broken open, releasing uncountable numbers of DNA fragments.

Over the years, they have built up a reference "library" or database of unique codes or "genetic signatures" from the DNA fragments in the samples. Each unique signature is a "fingerprint" or tag, representing a different kind of cell, explained Mike Rappé, postdoctoral researcher in Giovannoni’s lab.

"Molecular techniques allowed us to see the microbial diversity out there without seeing the organisms themselves," said Rappé.

"Studying open ocean bacteria is exciting," continued Rappé, who has helped to describe several new groups of ocean bacteria. "It is like big game hunting. You go out there into the ocean and it is like a jungle where you don’t know the names of anything. You don’t know what you’ll find. There could be a new discovery every day. It is a great field to be in." One by one, they have matched up genetic signatures of cells in samples with the codes of the organisms in their database and have given each type of cell they have found a code name, based on where it was sampled.

Morris, left, works with Giovannoni to remove water from a metal sampling canister they lowered into the Pacific Ocean. Back on the OSU campus, they'll try to isolate, reproduce and study microbes in the water sample to learn about their role in ocean ecosystems. Photo: Lynn Ketchum

A certain genetic signature, that of SAR number 11 or SAR11, kept showing up in incredible abundance in almost every sample Giovannoni and his team collected seawater from over the years. On average, SAR11 accounted for at least a quarter of the DNA fragments present in any given sample, regardless of where that sample was from.

After sampling upper layers of ocean water from all over the world, Giovannoni came to realize that they might have discovered one of the most abundant life forms on the planet.

Though SAR11 and its relatives may potentially account for a quarter or more of all the biomass in water, making it the most prevalent life form on Earth, researchers still have much to learn.

All they know is that SAR11 has a certain genetic signature, is very tiny compared to most bacteria and is ubiquitous and superabundant in the upper layers of the world’s open oceans as well as some freshwater bodies, including Crater Lake.

"These microorganisms, by virtue of sheer numbers, have great power out there in global ecosystems," said Giovannoni. "They play a major role in carbon cycling, which influences the atmospheric CO2 balance and probably climate."

A slide of common ocean bacteria called SAR 11 magnified 1,000 times with a florescent microscope. The colors come from varying DNA techniques used to label the organisms.

Giovannoni and his colleagues realize that they need much more than a genetic signature for each type of microbe they discover to truly understand the role of these microbes in the world’s oceans.

"Can we afford to not understand what microorganisms do?" he asked rhetorically. "Can we permit them to remain a mystery? Without them, there could be no life on Earth, no plant life, no animal life."

But there’s a big catch. To know what role they play, what and how much they consume, excrete and recycle, researchers must be able to grow the cells in culture. And these bacteria, by their nature are the toughest of the tough to grow in the lab.

"That’s the real challenge, making the jump from the genetic to the cellular level," he said. "Something about these bacteria makes them unable to grow in the lab using techniques we now have."

Back in his OSU lab, Steve Giovannoni works with a flow cytometer. Lasers in the device separate various microorganisms in a water sample collected about 15 miles off the Oregon Coast. Photo: Lynn Ketchum

For the past three years, Giovannoni and his research team have been designing, building and testing new ways to isolate and culture newly discovered, difficult-to-culture microbes such as SAR11 in the Laboratory for the Isolation of Novel Species in Nash Hall at OSU. The National Science Foundation, the Murdock Charitable Trust, Oregon Sea Grant, the OSU Agricultural Experiment Station and Diversa, a private biotechnology company, support these efforts.

Biotechnology companies interested in discovering new drugs work with research teams like Giovannoni’s in hope that the new and novel species of bacteria will be a source of new compounds and enzymes for medicine and agriculture.

With so many microbes to test and so many ways to try and grow them, the group has designed and built a unique "high-throughput" computer-controlled, robotics-assisted system for rearing and identifying microbes. Finely calibrated computer-driven machines mix and pipette seawater samples into super-dilute growth medium in sterile mini-test tubes. Racks of tubes, each seeded with a few seawater organisms, are incubated, filtered and checked for cell growth. Laser scanning cytometers detect the presence of life and determine an organism’s identity.

Giovannoni is visibly proud of his facility. "It’s my first foray into engineering," he says with a grin. "It’s the only high throughput system for raising microbes that I know of in the world."

And though it is early on in the project, they have already had some success in culturing elusive ocean bacteria. They have been able to culture dozens of types of microbes that have never been cultured in the laboratory before. Once they get a microbe to grow in culture, they quick-freeze it and store it in liquid nitrogen for future study or exchange with other scientists.

"With these techniques, we are able to successfully culture about 1 per 1,000 to 1 out of 10 ocean microbes we have tested, which is much better than the 1 per 10,000 to 100,000 that we could culture with traditional techniques," said postdoctoral researcher Mike Rappé.

But have they ever raised the ubiquitous SAR11?

"We once had it, and saw it under a microscope, said Giovannoni. "But when we went back to the culture tube, it was gone. But someday, I’m sure that we’ll culture it. And once we do, we may gain a huge understanding about global processes."

BOFFO BUGS BOOST BUSINESS

Recent advances in microbiology have spawned a major new industry called "biological prospecting," where industries are looking for microbial characteristics and activities that are useful.

"Microbes contain pathways and enzymes that catalyze reactions that can be used for commercial purposes, especially in medicine and agriculture," said OSU microbiologist Steve Giovannoni.

Illustration: Bill Lanham

"Microbial biodiversity is now regarded as a big resource by businesses," said Giovannoni. "Industry is learning to apply to commercial problems what microbiologists are discovering. Oftentimes, these biological pathways prove to be more efficient, cleaner, cheaper and more environmentally friendly than the traditional manufacturing process."

Have you noticed that laundry detergents have become more efficient over the years? You can thank microbes and their metabolic pathways.

"Detergents now often contain enzymes that can work to cut up proteins and hydrolyze lipids, even in cold water, using less detergent," explained Giovannoni. "These enzymes come from microbes."

Polymerase chain reaction, or PCR, a process used extensively today in criminal cases (e.g., O.J. Simpson), paternity suits and in screening for genetic traits was made possible because of an enzyme called Taq polymerase, discovered and extracted from a hot springs bacteria in Yellowstone National Park. Taq polymerase works at extremely high temperatures, making the PCR process fast and practical for automation. PCR is also used extensively in microbe studies such as Giovannoni’s.

"In the future, a lot more industries will use microbial processes," he said. "There will be microbial cells made that are highly engineered to do specific things, like make ascorbic acid, or vitamin C, from cheap, readily available carbon sources such as methane.

"And products from microbes will become a lot more important. Think of penicillin, a natural product of a microbial fungus. There is potential for lots of new natural products being discovered and tested to treat problems like arthritis."